CN114262365A

CN114262365A - Design of broad-spectrum rabies virus-like particle antigen and stable expression cell strain HEK-293 thereof

Info

Publication number: CN114262365A
Application number: CN202111469929.5A
Authority: CN
Inventors: 马兴元; 赵章婷; 郑文云; 邓昌平
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-04-01

Abstract

The invention firstly uses RVGP of CVS rabies virus RABV and RVLPs self-assembled by matrix protein RVMP as antigen to design a broad-spectrum rabies virus-like particle antigen; then the RVGP, the RVMP and the EGFP are jointly constructed into a eukaryotic expression vector pcDNA3.1(+), which is named pcDNA3.1(+) -RVLPs-EGFP. Each protein has an independent promoter (CMV), ribosome binding site (Kozak sequence) and PloyA tail (PA) so as to ensure the surface level of each protein, and EGFP is utilized to monitor the expression of the target protein in real time to construct eukaryotic recombinant expression plasmid; then transfecting the eukaryotic recombinant expression plasmid into HEK-293 cells, amplifying and passaging the cells, screening and identifying, and screening to obtain the positive monoclonal cell strain HEK-293/RVLPs stably and efficiently expressing the RVLPs. The obtained antigen has post-translational modification of mammalian cells, overcomes the defect that RVLPs from bacteria, yeast, insects and plant cells show low immunogenicity due to incorrect glycosylation modification, and lays a foundation for the large-scale production of the RVLPs antigen.

Description

Design of broad-spectrum rabies virus-like particle antigen and stable expression cell strain HEK-293 thereof

Technical Field

The invention belongs to the technical field of vaccines, and particularly relates to design of broad-spectrum rabies virus-like particles (RVLPs) antigen and screening and identification of a stable expression cell strain HEK-293 thereof.

Background

Rabies (Rabes) is an ancient and most easily overlooked human-animal co-disease, primarily triggered by the lethal pathogen Rabies Virus (Rabes Virus, RABV) (ZHao R Q, Shan Y, Li M H, et al. novel syndrome for Expression and Characterization of Rabes Viruses [ J ]. Protein Expression and Purification,2020,168:105567.Liu, ZHao W, W T, et al. Generation of Monoclonal Antibodies against Virus Variable Epitopes of the M Protein of Rabes Viruses [ J ]. Viruses,2019,11 (4)). Canines are the predominant host of RABV, and more than 99% of human Rabies is mediated by dogs (Navid M T, Li YY, Zhou M, et al. Complex of the Immunogenicity of the immunological conjugates of Two Inactivated microorganisms Viruses [ J ]. Archives of Virology,2016,161(10):2863-2870.), where RABV in saliva diffuses synaptically to peripheral nerves through the skin or mucosa at the wound and travels back to the Central Nervous System (CNS), replicates rapidly in the brain and causes brain damage with an apoptosis rate of almost 100% (Johnson N, Cunningham A F, photographs A R. factor to Viruses [ 21J.: 21, 23, 3906).

RABV is a strong neurotropic negative strand RNA virus belonging to the Rhabdoviridae (Rhabdoviridae family) RABV genus (Lyssavir gene). The genome of RABV (about 12 kb) consists of an unfragmented single-stranded negative-strand RNA encoding, in sequence, RVNP, RVPP, RVMP, RVGP, and RVLP 5 structural proteins (Navid M T, Li Y, Zhou M, et al. Complex of the oncogenesis of Two Inactivated Recombinant Viruses expressing the glycogene [ J ]. enzymes of Virology,2016,161(10):2863 and 2870.). RVGP is the only antigen exposed on the surface of the ravv that induces the body to produce Virus Neutralizing Antibodies (VNA). RVMP localizes Ribonucleoprotein complexes (RNPs) to the plasma membrane and binds RVGP to budding RABV particles, participating in the overall process of budding, replication and bullet formation of RABV from the bud (Liu, Zhao W, He W T, et al. RVMP self-assembles with RVGP into Virus Like Particle (VLP) structures even in the absence of other components of the RABV.

Although rabies is a vaccine preventable disease, it still causes about 6 million deaths per year worldwide, with children accounting for 40-50% [5 ]. Due to the defects of the existing vaccine in the aspects of immune effect and the like, multiple times of administration are needed, and the cost benefit needs to be improved.

VLPs are Nanoparticles (NPs) formed by self-assembly of one or several structural proteins of a virus, without nucleic acids, similar in morphology and structure to the original virus. VLPs act as multiprotein supramolecular structures containing viral key antigens, displaying high density epitopes on their surface in a repetitive fashion, facilitating cross-linking of B-lymphocyte receptors (BCRs), and can induce a strong B-cell response (Rybicki E P. plant Molecular proofing of viruses-Like nanoparticules as Vaccines and Reagents [ J ]. Wiley Interdisc review-Nanomedicine and Nanobiotechnology,2020,12(2): E1587.). Furthermore, the particle structure of VLPs can facilitate the uptake of Antigen Presenting Cells (APCs), stimulate innate and adaptive immune responses (Ludwig C, Wagner R.Virus-Like Particles-Universal Molecular weights [ J ]. Current Opinion in Biotechnology,2007,18(6):537 545.).

Many studies have shown that RVGP expressed by yeast, insect and plant cells as antigen does not provide adequate protection (one H K, Tan W S, Ho K L. Virus Particles as a Platform for Cancer Vaccine Development [ J ]. PeerJ,2017,5: e4053.Zhang Y C, Zhou M, Li Y, et al. Binding reagents viruses with the carbohydrate Peptide in Fused Peptide salts with a DC-Binding Peptide Particles Vaccine [ J ]. Oncott, 2018,9(1):831-841.Wang S J, Liu H Q, Zhang X Y, Imal. internal and expression proteins-Antigens as well as Antigens [ J ]. expression J ]. 12. expression J.: modification and expression of proteins, expression J.: cells, expression J.: modification and expression J. (3. 12. expression J.), 2018,9:1432.Kato T, Deo V K, Park E Y.functional Virus-Like Particles Production Using Silkworks and the ir Application in Life Science [ J ]. Journal of Biotechnology & Biomaterials,2012, S9(1):1-7.Scott N, Rybicki E P.Virus-Like Particles Production in Plants As positional variables [ J ]. Ext Review of variables, 2013,12(2):211 224.), apparently due to a different glycosylation structure than that of eukaryotic cells. Currently, much research is devoted to the production of VLP vaccines containing RVGP by infecting HEK-293 cells with viral vectors, which can protect mice from viral challenge and enable large-scale production. However, such vaccines may have infectious viral contamination with the vector.

Disclosure of Invention

The present invention has been made in view of the above problems, and has been made in an effort to develop RVLPs having sufficient safety and broad spectrum, and to obtain a stable cell line HEK-293 by screening in order to realize mass production of the RVLPs.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention firstly utilizes RVLPs (Rabies virus proteins, RVGP) self-assembled by glycoprotein (RBIS virus proteins, RVMP) and matrix protein (RBIS virus matrix proteins, RVMP) of CVS strain Rabies virus (RABV) as antigen to design a broad-spectrum Rabies virus sample particle antigen; then the RVGP, the RVMP and the EGFP are jointly constructed into a eukaryotic expression vector pcDNA3.1(+), which is named pcDNA3.1(+) -RVLPs-EGFP. Each protein has an independent promoter (CMV), ribosome binding site (Kozak sequence) and PloyA tail (PA) to ensure the surface level of each protein, and EGFP is used for monitoring the expression of the target protein in real time to construct eukaryotic recombinant expression plasmid; then transfecting the eukaryotic recombinant expression plasmid into HEK-293 cells, amplifying and passaging the cells, screening and identifying, and screening to obtain the positive monoclonal cell strain HEK-293/RVLPs stably and efficiently expressing the RVLPs. The obtained antigen has post-translational modification of mammalian cells, overcomes the defect that RVLPs from bacteria, yeast, insects and plant cells show low immunogenicity due to incorrect glycosylation modification, and lays a foundation for the large-scale production of the RVLPs antigen.

In a first aspect of the invention, a broad-spectrum rabies virus-like particle antigen is provided, which comprises a CVS strain rabies virus glycoprotein RVGP and a matrix protein RVMP. The polynucleotide sequence of the CVS rabies virus glycoprotein RVGP which is optimized by the codon is shown as SEQ ID NO.1, and the polynucleotide sequence of the matrix protein RVMP which is optimized by the codon is shown as SEQ ID NO. 2.

Preferably, the CVS strain rabies virus glycoprotein RVGP has 4 possible N-glycosylation sites Asn56, Asn266, Asn338 and Asn484, the first 3 being located in the ectodomain of RVGP, the 4 th being located in the cytoplasmic tail of RVGP; the ectodomain of the CVS strain rabies virus glycoprotein RVGP contains B-cell epitopes, CLT-cell epitopes and Th-cell epitopes, and Asn56 and Asn266 are contained in the B-cell epitopes and Asn338 is contained in the CTL-cell epitopes.

The design method of the broad-spectrum rabies virus-like particle antigen comprises the following steps:

(1) the protein sequences of the CSV strains RVGP and RVMP were obtained using the on-line website NCBI (https:// www.ncbi.nlm.nih.gov /).

(2) B cell epitopes in the RVGP antigen sequence are predicted by the online software ABCPred prediction server (http:// crdd. osdd. net/raghava/ABCpred /), helping to locate epitope regions which can be used to identify candidate vaccines.

(3) Potential CTL epitopes in RVGP were predicted by the online software bioxgem. pacomplex (http:// pacomplex. life. nctu. edu. tw. /) and SYFPEITHI (http:// www.syfpeithi.de/bin/mhcserver. dll/findyourmotif. htm).

(4) Potential Th epitopes in RVGP were predicted by the on-line software RANKPEP (https:// omictools. com/RANKPEP-tool).

(5) The N-glycosylation sites in the RVGP were predicted by the online software NetNGlyc 1.0Server (http:// www.cbs.dtu.dk/services/NetNGlyc /).

(6) Epitope and tertiary structure changes of RVGP after glycosylation site mutation were analyzed by bioinformatics software and PyMOL software in (2) - (4).

(7) Obtaining 34 RVGP amino acid sequences separated from different regions in China from NCBI, comparing the obtained RVGP with the RVGP of a CSV strain by using DNAMAN software, determining the sequence consistency of the candidate RVGP and the RVGP of China origin, and further judging whether the candidate RVGP can be used as a candidate antigen of the ORV for Chinese animals.

In a second aspect of the invention, there is provided a eukaryotic recombinant expression plasmid comprising the broad-spectrum rabies virus-like particle antigen described above.

The construction method of the eukaryotic recombinant expression plasmid comprises the following steps:

1) respectively amplifying a promoter (CMV), an SV40PA and a green fluorescent protein EGFP by taking a eukaryotic plasmid pcDNA3.1(+) as a template;

2) performing overlap extension PCR amplification on target fragments SV40PA-CMV and SV40PA-CMV by using a promoter and SV40PA as templates;

3) and performing overlap extension PCR amplification on a target fragment SV40PA-CMV-EGFP by using SV40PA-CMV and a fluorescent protein as templates.

Preferably, in eukaryotic recombinant expression plasmids, RVGP, RVMP and fluorescent protein all have separate promoters (CMV), ribosome binding site (Kozak sequence) and PloyA tail (PA).

In a third aspect of the invention, a eukaryotic expression system for broad-spectrum rabies virus-like particle antigen is provided, wherein the eukaryotic expression system is HEK-293 cells or other mammalian cells capable of realizing post-translational modification of mammalian carbohydrates.

The method for screening and identifying the HEK-293 cell stably expressing the broad-spectrum rabies virus-like particle antigen comprises the following steps:

1) transfecting a eukaryotic plasmid SV40PA-CMV-EGFP into HEK-293 cells, and culturing the cells for screening cell strains stably expressing RVLPs;

2) protein and mRNA levels of RVLPs were measured 48h after transfection by WB and qPCR;

3) adopting 2mg/mL G418 to screen HEK-293 cell strain stably expressing broad-spectrum rabies virus-like particle antigen, and realizing amplification and passage of positive clone strain after culture and enrichment;

4) digesting the positive monoclonal cell strain, detecting the proportion and the average fluorescence intensity of EGFP positive cells by using a flow method, detecting the expression condition of RVLPs by using WB, selecting the cell strain with the EGFP positive cell proportion of more than 99 percent as a candidate stable cell strain, optimally selecting the cell strain with the highest average fluorescence intensity and RVLPs expression quantity as the stable cell strain, and naming the cell strain as HEK-293/RVLPs;

5) subjecting HEK-293/RVLPs to subculture, digesting the 10 th generation cells, inoculating a part of HEK-293/RVLPs into a confocal culture dish for culture, fixing the HEK-293/RVLPs with 4% paraformaldehyde at room temperature after 12h, staining nuclei for 10min with Hoechest 33342, washing with PBS, and observing the proportion of EGFP positive cells by using a laser confocal microscope; a part of cells are used for flow detection of EGFP positive cell proportion; inoculating a part of cells into a 24-well plate at different densities, and identifying the expression level of the target protein by WB; simultaneously collecting HEK-293/RVLPs cells for cell ultrathin section; collecting culture medium, extracting total cell protein of HEK-293/RVLPs, and performing sucrose density gradient centrifugation to realize identification and purification of RVLPs.

Preferably, 48h after transfection, the transfection efficiency is measured by flow cytometry, and then step (2) is performed to measure protein and mRNA levels of RVLPs using WB and qPCR.

Preferably, step 2) may be temporarily omitted 48 hours after transfection, and 2mg/mL of G418 is selected based on the result of MTT to select stable cell lines, and the optimal concentration of G418 is 2.235. + -. 0.206mg/mL, followed by step 3.

In the step 3), when the positive monoclonal cell strain is cultured, replacing a fresh DMEM medium containing 2mg/mL G418 once every 2 days for 14 days; when the positive monoclonal cell strain is enriched, cells are digested by pancreatin and diluted to 1/200 mu L, 200 mu L of the mixture is uniformly mixed and added into a 96-well plate, a fresh DMEM medium containing 1mg/mL is replaced once every 3 days, and the positive monoclonal cell strain is enriched for 7 days.

Preferably, in step 5), when the cell superfabric section is detected, the method is as follows:

digesting and collecting cell sediment with the size of mung bean; washing with PBS for 3 times, and fixing with 2.5% paraformaldehyde at 4 deg.C overnight;

② the fixed cells are embedded in epoxy resin after dehydration treatment by ethanol solution with increasing concentration. Carrying out ultrathin cell slicing in a galvano-mirror chamber and fixing the ultrathin cell slice on a copper net;

③ after the negative staining of 1 percent phosphotungstic acid for 20 to 30s, observing the location of the RVLPs in the cells under a Transmission Electron Microscope (TEM) (with the voltage of 200 kV).

The procedure for identifying and purifying RVLPs was as follows:

harvesting and culturing HEK-293/RVLPs cell culture medium, filtering with 0.45 mu m filter membrane, concentrating with 30KDa ultrafiltration tube, and storing the concentrated supernatant at-80 ℃;

② extracting the total protein of the HEK-293/RVLPs cell;

thirdly, centrifuging the supernatant of the first step and the second step for 3 hours at 4 ℃ and 30000rpm, and dissolving the precipitate with PBS overnight at 4 ℃;

fourthly, preparing sucrose solutions with the concentration of 20 percent, 40 percent and 60 percent, and filtering the sucrose solutions by a filter membrane with the diameter of 0.45 mu m;

adding the sucrose solution into an ultracentrifuge tube in sequence from low to high to form a density gradient of 20-40-60%, adding the dissolved centrifugal product, and centrifuging at 35000rpm for 2h at 4 ℃;

sixthly, collecting white bands positioned between 40 percent and 60 percent of sucrose layers, centrifuging the white bands in PBS at 4 ℃ and 30000rpm for 3 hours for desugarization treatment, and dissolving the precipitate with PBS overnight at 4 ℃;

seventhly, the BCA kit is used for measuring the concentration of the RVLPs, and the RVLPs are stored at the temperature of minus 80 ℃ for later use;

allowing WB to identify RVLPs proteins among different sucrose layers;

ninthly, dropwise adding 10 mu L of the purified RVLPs sample on a copper net, standing for 15-20min, sucking residual liquid by using filter paper, negatively dyeing by 1% phosphotungstic acid for 20-30s, sucking off redundant liquid, standing until the copper net is dried, and observing the sample by using a TEM (voltage of 120 kV).

The invention has the following beneficial effects:

antigen aspect: the invention assembles RVGP and RVMP from CSV strains to obtain RVLPs which are used as broad-spectrum antigens for preventing Chinese canine rabies. The results of bioinformatics software analysis show that the ectodomain of RVGP contains all antigen epitopes and 3 main N-glycosylation sites; mutations in the N-glycosylation site of the ectodomain result in loss of the RVGP spatial epitope and structural changes; the consistency of the candidate RVGP and the RVGP sequence of 34 strains derived from other regions in China reaches 97.25%, and the main antigen binding site and all glycosylation sites are highly conserved, which indicates that the selected RVGP can be used as the candidate antigen for the rabies in China.

Selection of cell lines: the invention obtains the positive monoclonal cell strain HEK-293/RVLPs which stably and efficiently express the RVLPs by screening by transfecting eukaryotic plasmids containing the RVGP and the RVMP to HEK-293 cells. WB and TEM results show that RVLPs can be detected in cells and in culture medium, and are separated by 20-40-60% sucrose density gradient centrifugation, the separated RVLPs are mainly positioned in 40-60% sucrose layers and are in atypical bullet-shaped forms, but are in circular or oval structures, and analysis is possible because natural RVLPs contain RNPs complexes besides RVMP and RVGP, and the RNPs are closely connected and play a certain supporting role in the RVLPs.

Therefore, the RVLPs constructed by the present invention lack other internal elements and have no supporting function, so that the RVLPs are morphologically different from natural RVLPs. The HEK-293 cell is used as an expression system, has a perfect PTM mechanism, and can ensure that the RVGP obtains a correct glycosylation structure, so that the immunogenicity of the RVGP is ensured. The constructed HEK-293/RVLPs stable cell strain lays a foundation for producing high-immunogenicity RVLPs.

Drawings

Figure 1 is a sequence analysis and alignment of candidate RVGP.

FIG. 2 is a prediction of the N-glycosylation site of the RVGP of the CSV strain;

FIG. 3 shows the results of RVGP epitope and N-glycosylation site analysis;

FIG. 4 is a graph of the effect of N-glycosylation sites on RVGP antigenicity and tertiary structure;

FIG. 5 shows the result of comparing the RVGP protein sequences of 34 strains of Chinese rabies virus and CVS strain;

FIG. 6 is an electrophoretogram constructed from pcDNA3.1(+) -RVLPs-EGFP;

FIG. 7 shows the construction of pcDNA3.1(+) -RVLPs-EGFP and the process of obtaining HEK-293 cell lines stably expressing RVLPs.

FIG. 8 shows that pcDNA3.1(+) -RVLPs-EGFP plasmid transfects HEK-293 cells: wherein, (A) a fluorescence map; (B) transfection efficiency.

FIG. 9 shows RVLPs (A) mRNA and (B) protein levels in HEK-293 cells;

FIG. 10 is a graph of the screening and enrichment of HEK-293/RVLPs positive cell lines, in which: (A) transfection efficiency of HEK-293 cells; (B) the cell survival rate and the IC50 value of HEK-293 cells after 48 hours of action of G418 with different concentrations; (C) morphological changes and fluorescence distribution of HEK-293 cells treated with 2mg/mL G418.

FIG. 11 is an identification of positive monoclonal cell strains of HEK-293/RVLPs, wherein: (A) the proportion of different positive clonal cells; (B) mean fluorescence intensity and (C) protein level of RVLPs.

FIG. 12 is the generation stability of HEK-293/RVLPs cell line, in which: (A) CLSM representation of HEK-293/RVLPs cell lines; (B) EGFP levels in HEK293/RVLPs cells; (C) expression levels of RVLPs in HEK-293/RVLPs cells of different confluency.

FIG. 13 is a representation of RVLPs wherein: (A) TEM observation RVLPs distribution in cells and medium; (B) purifying the RVLPs by sucrose density gradient centrifugation; (C) WB analysis sucrose density gradient centrifugation samples.

Detailed Description

The following embodiments are implemented on the premise of the technical scheme of the invention, and give detailed implementation modes and specific operation procedures, but the protection scope of the invention is not limited to the following embodiments.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Example 1 broad-spectrum rabies virus-like particle (RVLPs) antigen design

Sequence analysis of RVGP

The nucleotide sequences of the CSV strains RVGP and RVMP are obtained from NCBI database and are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively. The sequence analysis and alignment results of the candidate RVGP are shown in figure 1.

The B cell epitopes (table 1), CLT cell epitopes and Th cell epitopes (table 2) of the resulting RVGP were all located in the ectodomain of mature RVGP as determined by bioinformatics software.

TABLE 1B-cell epitope analysis of the CSV Strain RVGP

TABLE 2T-cell epitope analysis of the CSV strain RVGP

Prediction of RVGP glycosylation sites

The mature RVGP membrane ectodomain has 3 potential N-glycosylation sites (Asn37, Asn247, and Asn319), i.e., Asn56, Asn266, and Asn338 sites of the immature RVGP. N-glycosylation affects the antigenicity and correct folding of RVGP, and RVGP with the correct N-glycosylation structure can be produced by means of eukaryotic expression systems.

Candidate RVGP was analytically determined to have 4 possible N-glycosylation sites (Asn56, Asn266, Asn338 and Asn484) (figure 2). By integrating the predicted epitope together with the glycosylation site into the protein sequence of RVGP, it can be found that the ectodomain of RVGP contains all epitopes and that the first 3 of the predicted 4N-glycosylation sites are located in the ectodomain of RVGP (Asn56, Asn266 and Asn338) and the 4 th is located in the cytoplasmic tail of RVGP (Asn 484). Among them, Asn56 and Asn266 were contained in the B-cell epitope and Asn338 in the CTL epitope, indicating that the ectodomain is the critical region for RVGP to function as an antigen (fig. 3).

3. Effect of glycosylation sites on the antigenicity and spatial structure of RVGP

The correct glycosylation structure is crucial for RVGP to exert an immune function. When Asn56 mutated, the spatial B-cell epitope at that position was lost (fig. 4A), while the secondary structure at that position was mutated from a β -sheet to a coil (fig. 4B); whereas mutations in the glycosylation site had little effect on the linear epitope (FIG. 4A), the spatial structure of RVGP was not significantly altered when Asn266 and Asn338, which are located in linear B-cell and CTL epitopes, were mutated (FIG. 4B). The results show that the N-glycosylation sites influence the spatial structure and antigenicity of the RVGP, and the selection of an expression system capable of expressing the RVGP with the correct N-glycosylation structure plays a key role in the development of rabies vaccines.

RVGP amino acid sequence identity analysis

The sequence consistency of the RVGP sequences and the RVGP sequences of the CVS strains is analyzed by using DNAMAN software, the analysis result shows that the sequence consistency reaches 97.25 percent, and the key antigenic sites and glycosylation sites are highly conserved, which indicates that the RVGP of the CVS strains can be used as candidate antigens of the rabies vaccine in China (figure 5).

Example 2 construction of recombinant plasmid pcDNA3.1(+) -RVLPs-EGFP

The whole gene synthesized RVGP and RVMP were constructed into pcDNA3.1 (+). CMV, SV40PA and EGFP are amplified respectively by taking pcDNA3.1(+) as a template; then, overlapping and extending the target fragment (G) SV40PA-CMV and (M) SV40PA-CMV by using CMV and SV40PA as templates; finally, the target fragment (G) SV40PA-CMV-EGFP is amplified by overlap extension PCR by taking (G) SV40PA-CMV and EGFP as templates.

The electrophoresis result of the obtained target fragment is shown in FIG. 6, and is consistent with the actual size (CMV:618 bp; SV40 PA:122 bp; (G)/(M) SV40 PA-CMV:760 bp; EGFP:759 bp; (G) SV40 PA-CMV-EGFP:1484bp), which indicates the success of construction.

Example 3 construction of HEK-293 eukaryotic expression System

1. Eukaryotic expression system construction process

FIG. 7 shows the construction of pcDNA3.1(+) -RVLPs-EGFP and the process of obtaining HEK-293 cell lines stably expressing RVLPs, which is as follows:

2. Detection of transfection efficiency of HEK-293 cells

The optimal transfection mode is confirmed by transfecting HEK-293 cells with pcDNA3.1(+) -RVLPs-EGFP by using PEI and liposome reagents respectively. 48h after transfection, the transfection was first qualitatively observed by inverted fluorescence microscopy (FIG. 8A), and then the transfection efficiency was quantitatively analyzed by FCM (FIG. 8B). The results show that the transfection efficiency of both reagents reaches more than 60%, when the DNA: PEI 1: 4, transfection efficiency was highest (65.7%), followed by liposomes (65.1%), and liposome cytotoxicity was less than PEI, so liposomes were used as transfection reagents to screen HEK-293 stable cell lines overexpressing RVLPs (figure 8).

3. Detection of RVLPs

Collecting HEK-293 cells and culture media transfected by pcDNA3.1(+) -RVLPs-EGFP for 48h, detecting the mRNA level of the RVLPs in the cells by utilizing qPCR, and detecting the protein level of the RVLPs in the cells and the culture media by utilizing WB, wherein the specific method comprises the following steps:

(1) WB assay for RVLPs protein levels

Concentrating a cell culture medium by using a 30KDa ultrafiltration tube;

② after the cells are digested by pancreatin, the cells are collected by centrifugation for 5min at 1000 rpm.

③ after washing 3 times with PBS, RIPA cell lysate containing 1% PMSF was added and lysed at 4 ℃ for 30 min.

Fourthly, centrifuging for 10min at the temperature of 4 ℃ and the rpm of 12000, and sucking the supernatant fluid to store at the temperature of minus 80 ℃.

Using BCA method to determine the protein concentration in the culture medium and cell lysis supernatant, and carrying out SDS-PAGE with the sample loading amount of 30 mu g.

Cutting gel of the target protein, cutting PVDF membrane with corresponding size, and activating in anhydrous methanol for 5 min;

seventhly, sequentially stacking the sponge, the 3 layers of filter paper, the gel, the PVDF membrane, the 3 layers of filter paper and the sponge soaked by the membrane transferring buffer solution on the cathode plate, fastening the anode plate, putting the cathode plate into a membrane transferring device, and electrically transferring for 1.5 hours at the temperature of 4 ℃ and under the voltage of 200 mA.

Eighthly, after 1.5h, taking out the PVDF membrane, sealing the PVDF membrane by using 3 percent skimmed milk powder at room temperature for 2h, and then washing the PVDF membrane by using TBST for 3 times and 5 min/time;

ninthly, respectively mixing the sealing liquid with the following components in percentage by weight: 200 and 1: diluting mouse anti-RVGP monoclonal antibody and rabbit anti-RVMP polyclonal antibody at the ratio of 500, shaking with PVDF membrane at 4 ℃ overnight, and washing the membrane with TBST for 3 times and 5 min/time;

sealing liquid for r 1: diluting HRP-labeled goat anti-rabbit/mouse IgG 6000 times, and incubating with a PVDF membrane at room temperature for 1h in a shaking manner; washed 3 times with TBST for 10 min/time, and then ECL chemiluminescence imaged.

(2) qPCR detection of mRNA levels of RVLPs

The qPCR primer sequences are shown in Table 3.

② primer dilution to 10 μ M, according to the table 4 on ice to prepare qPCR reaction solution, shaking to fully mix, centrifuging to carry out qPCR reaction.

③ using a Bio-Rad CFX96 qPCR instrument to carry out reaction, and the reaction program is briefly described as follows: pre-denaturation: 95 ℃ for 5 s; and (3) PCR reaction: [95 ℃,5 s; 55 ℃ for 10 s; the dissolution curve was obtained at 72 ℃ for 20 s.times.40 cycles.

And fourthly, analyzing the expression level of the target gene by adopting a 2-delta Ct result of the qPCR. The formula is as follows: the expression level is 2- Δ Δ Ct, where Δ Δ Ct is [ (Ctgene in test cell line-Ctgene in control cell line) - (Ct β -actin in test cell line-Ct β -actin in control cell line) ].

TABLE 3 qPCR primer List

TABLE 4 qPCR reaction System

As can be seen from FIG. 9, the transcript levels of RVGP and RVMP were essentially identical, and both RVGP and RVMP proteins were detectable in HEK-293 cells and in the culture medium. Since the volume of the medium after the medium concentration was 100 times that of the cell lysis supernatant, it was revealed that the RVLPs assembled from RVGP and RVMP were mostly secreted into the medium.

4. Screening and identification of positive monoclonal cell strain HEK-293/RVLPs

MTT experimental results showed that the IC50 of G418 to HEK-293 cells was 2.235. + -. 0.206mg/mL (FIG. 10B). Therefore, 2mg/mL G418 was selected for the screening of HEK-293/RVLPs cell lines. After HEK-293 cells were transfected with pcDNA3.1(+) -RVLPs-EGFP for 48h, one group of cells was tested with FCM and the transfection efficiency was 65.66% (FIG. 10A). Another group of cells was continuously screened with DMEM medium containing 2mg/mL G418 for 14 days to enrich positive cells (FIG. 10C). Then, positive monoclonal cells were amplified in a 96-well plate by limiting dilution, and a cell line having an EGFP-positive cell ratio of more than 99% was selected by FCM and named HEK-293/RVLPs clone 1/2/4/5/8/10/11/12 (FIG. 11A). FCM and WB results showed that mean fluorescence intensity and protein expression levels of clone 11 were significantly higher than other positive monoclonal cell lines (P <0.05) (fig. 11B and C). Thus, clone 11 will be used in subsequent studies and will be named HEK-293/RVLPs.

5. Passage stability of HEK-293/RVLPs cell line

And transferring the obtained RVLPs high-efficiency expression cell strain HEK-293/RVLPs to the 10 th generation, digesting and inoculating the cell strain into a copolymerization focal culture dish, fixing by 4% paraformaldehyde, carrying out nuclear staining by using Hoecst 33342 dye, and carrying out CLSM observation and photographing. CLSM results showed that each cell expressed EGFP and was mainly localized to the cytoplasm (fig. 12A). FCM results showed that the proportion of EGFP-positive cells was 100% (fig. 12B). The remaining cells were cultured and inverted fluorescence microscopy showed no loss of green fluorescence in the cells, WB detected HEK-293/RVLPs cell protein expression levels at different confluency, indicating no decrease in RVLPs intracellular expression levels (FIG. 12C).

6. Characterization of RVLPs

Pancreatin digestion harvest HEK-293/RVLPs cells in T25 flasks were subjected to ultrathin cell sectioning as follows:

Meanwhile, TEM is used for observing the RVLPs purified by sucrose density gradient centrifugation, and the method is as follows:

② extracting the total protein of the HEK-293/RVLPs cell;

allowing WB to identify RVLPs proteins among different sucrose layers;

As shown in the left panel of 13A, RVLPs with the size of between 180-200nm are observed in the vesicle and outside the cell of the HEK-293/RVLPs, which indicates that the RVLPs can be secreted into the culture medium after being expressed; there are round or oval particles with sizes between 180-200nm (right in FIG. 13A). The RVLPs were predominantly in the 40% -60% sucrose layer after density gradient centrifugation (FIG. 13B), and the results of WB fully illustrate that the NPs observed were self-assembled from RVGP and RVMP into RVLPs (FIG. 13C). Although the self-assembled RVLPs have different sedimentation coefficients, most of the RVLPs are located in 40-60% of the sucrose layer, which indicates that most of the RVLPs are self-assembled according to the assembly proportion of the virus.

In conclusion, the RVLPs assembled by the CSV strains RVGP and RVMP are used as broad-spectrum antigens for preventing rabies of Chinese dogs. The results of bioinformatics software analysis show that the ectodomain of RVGP contains all antigen epitopes and 3 main N-glycosylation sites; secondly, mutations in the N-glycosylation site of the ectodomain lead to loss of the RVGP spatial epitope and structural changes; finally, the consistency of the candidate RVGP and the RVGP sequences of 34 RVGP strains from other regions in China reaches 97.25%, and the main antigen binding sites and all glycosylation sites are highly conserved, which indicates that the selected RVGP can be used as the candidate antigen for the rabies in China.

The invention obtains the positive monoclonal cell strain HEK-293/RVLPs which stably and efficiently express the RVLPs by screening by transfecting eukaryotic plasmids containing the RVGP and the RVMP to HEK-293 cells. WB and TEM results show that RVLPs can be detected in cells and in culture medium, and are separated by 20-40-60% sucrose density gradient centrifugation, the separated RVLPs are mainly positioned in 40-60% sucrose layer, are in atypical bullet-shaped forms and are in circular or oval structures, and the analysis is probably because the natural RVLPs also contain RNPs complexes inside in addition to RVMP and RVGP, and the RNPs are tightly connected and play a certain supporting role inside the RVLPs. The RVLPs constructed in the research lack other internal elements and have no supporting effect, so the RVLPs are morphologically different from natural RVLPs.

The HEK-293 cell is used as an expression system, has a perfect PTM mechanism, and can ensure that the RVGP obtains a correct glycosylation structure, so that the immunogenicity of the RVGP is ensured. To potentiate the immune effects of RVLPs, this chapter also constructs and expresses mucosal immune adjuvant LTB, which is able to bind to the GMI receptors on most cell surfaces, enhancing the humoral, cellular and mucosal immune responses of oral RVLPs. In a word, the HEK-293/RVLPs stable cell strain established by the method lays a foundation for producing high-immunogenicity RVLPs, and the immunogenicity of the RVLPs is further enhanced by using a mucosal adjuvant.

In the present invention, the coding sequence of codon-optimized RVGP (SEQ ID NO.1)

Wherein the content of the first and second substances,AAGCTTis a Hind III restriction enzyme site,

is a Kpn I restriction enzyme site,GCGGCCGCa Not I restriction enzyme site,

is a ribosome binding site and is a protein capable of binding to the ribosome,GTCGACis a restriction site for Sal I,

is a restriction site for BamHI,GAATTCis an EcoR I restriction site.

Codon optimized RVMP coding sequence (SEQ ID NO.2)

Wherein the content of the first and second substances,GCTAGCis a Nhe I restriction enzyme site;

is a ribosome binding site and is a protein capable of binding to the ribosome,AAGCTTHindIII restriction sites.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Sequence listing

<110> university of east China's college of science

<120> design of broad-spectrum rabies virus-like particle antigen and stable expression cell strain HEK-293 thereof

<130> claims, specification

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1619

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

aagcttggta ccgcggccgc gccaccatgg tgcctcaggc cctgctgttc gtgccactcc 60

tggtgttccc actgtgcttc ggcaagttcc caatttacac aattcctgac aagctgggcc 120

cttggtcacc tattgacatt caccacctct cttgccctaa caacctcgtg gtggaggacg 180

agggctgcac aaacctgtct ggcttctctt acatggagct gaaggtgggc tacattctcg 240

ccattaagat gaacggcttc acatgcacag gcgtggtgac agaggccgag acatacacaa 300

acttcgtggg ctacgtgacc accaccttca agcggaagca cttcagacct acccccgacg 360

cctgccgcgc cgcctacaac tggaagatgg ccggcgaccc tagatacgag gagtctctcc 420

acaaccctta ccccgactac cactggctca gaacagtcaa gacaacaaag gagtctctgg 480

tgattatcag cccatctgtg gccgacctcg acccttacga ccgcagcctc cactcgcgcg 540

tgttccctag cggcaagtgc ccaggcgtgg ccgtgtctag cacatactgc tctaccaacc 600

acgactacac catttggatg ccagagaacc ctagactcgg catgtcttgc gacattttca 660

ccaacagtag gggcaagcgc gcctctaagg gcagcgagac atgcggcttc gtggacgagc 720

ggggcctgta caagagcctg aagggcgcct gcaagctgaa gctgtgcggc gtgctcggcc 780

tgagactgat ggacggcaca tgggtggcca tgcagacatc taacgagaca aagtggtgcc 840

ctccagacca gctcgtgaac ctccacgact tccggtccga cgagattgag cacctcgtgg 900

tggaggagct ggtgagaaag cgcgaggagt gcctcgacgc cctagaaagc attatgacaa 960

caaagtctgt gtctttccgc agactgagcc acctgcggaa gctggtgcca ggcttcggca 1020

aggcctacac aattttcaac aagacactca tggaggccga cgcccactac aagtctgtga 1080

gaacatggaa cgagattctc ccatctaagg gctgcctcag ggtgggcggc agatgccacc 1140

cccacgtgaa cggcgtgttc ttcaacggca ttattctggg ccccgacggc aacgtgctca 1200

tcccagagat gcagtctagc ctgctccagc agcacatgga gctgctggaa tcctctgtga 1260

ttccactcgt gcacccactc gccgacccat ctacagtgtt caaggacggc gacgaggccg 1320

aggacttcgt ggaggtgcac ctccccgacg tgcacaacca ggtgtctggc gtggacctgg 1380

gcctcccaaa ctggggcaag tacgtgctcc tgtccgccgg cgccctcacc gccctgatgc 1440

tcattatttt cctgatgaca tgttgtagaa gagtgaatcg gtctgagcca acacagcaca 1500

acctgcgggg cacaggcagg gaggtgtccg tgacccctca gtccggcaag atcattagct 1560

cttgggagtc tcacaagtcc ggcggccaga ccagactgtg agtcgacgga tccgaattc 1619

<210> 2

<211> 627

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gctagcgcca ccatgaactt tctacgtaag atagtgaaaa attgcaggga cgaggacact 60

caaaaaccct ctcccgtgtc agcccctctg gatgacgatg acttgtggct tccaccccct 120

gaatacgtcc cgctgaaaga acttacaagc aagaagaaca tgaggaactt ttgtatcgac 180

ggaggggtta aagtgtgtag cccgaatggt tactcgttca ggatcctgcg gcacattctg 240

aaatcattcg acgagatata ttctgggaat cataggatga tcgggttagt caaagtagtt 300

attggactgg ctttgtcagg atctccagtc cctgagggca tgaactgggt atacaaattg 360

aggagaacct ttatcttcca gtgggctgat tccaggggcc ctcttgaagg ggaggagttg 420

gaatactctc aggagatcac ttgggatgat gatactgagt tcgtcggatt gcaaataaga 480

gtgattgcaa aacagtgtca tatccagggc agaatctggt gtatcaacat gaacccgaga 540

gcatgtcaac tatggtctga catgtctctt cagacacaaa ggtccgaaga ggacaaagat 600

tcctctctgc ttctagaata aaagctt 627

<210> 3

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

Asp Gly Thr Trp Val Ala Met Gln Thr Ser Asn Glu Thr Lys Trp Cys

1 5 10 15

<210> 4

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 4

Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys

1 5 10 15

<210> 5

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

Leu Gly Leu Arg Leu Met Asp Gly Thr Trp Val Ala Met Gln Thr Ser

1 5 10 15

<210> 6

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 6

Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Pro

1 5 10 15

<210> 7

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys Thr Leu Met Glu

1 5 10 15

<210> 8

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu His Asn Pro

1 5 10 15

<210> 9

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Ser Gly Lys Ile Ile Ser Ser Trp Glu Ser His Lys Ser Gly Gly Gln

1 5 10 15

<210> 10

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Gln Leu Val Asn Leu

1 5 10 15

<210> 11

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu

1 5 10 15

<210> 12

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr

1 5 10 15

<210> 13

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 13

Leu Arg Thr Val Lys Thr Thr Lys Glu Ser Leu Val Ile Ile Ser Pro

1 5 10 15

<210> 14

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala Ala

1 5 10 15

<210> 15

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

Thr Tyr Thr Asn Phe Val Gly Tyr Val

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 16

Ala Tyr Thr Ile Phe Asn Lys Thr Leu

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 17

Cys Pro Pro Asp Gln Leu Val Asn Leu

1 5

<210> 18

<211> 14

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 18

Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val Lys Thr Lys

1 5 10

<210> 19

<211> 15

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 19

Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu Ser Ile Met Thr

1 5 10 15

<210> 20

<211> 15

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 20

Leu Arg Val Gly Gly Arg Cys His Pro His Val Asn Gly Val Phe

1 5 10 15

<210> 21

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

ctgctctacc aaccacgact acac 24

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

gtccacgaag ccgcatgtct c 21

<210> 23

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

tggatgacga tgacttgtgg cttc 24

<210> 24

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

aacgagtaac cattcgggct acac 24

<210> 25

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

attggcaatg agcggttc 18

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

atactcctgc ttgctgatcc 20

Claims

1. The broad-spectrum rabies virus-like particle antigen is characterized by comprising a CVS strain rabies virus glycoprotein RVGP and a matrix protein RVMP, wherein the polynucleotide sequence of the CVS strain rabies virus glycoprotein RVGP after codon optimization is shown as SEQ ID NO.1, and the polynucleotide sequence of the matrix protein RVMP after codon optimization is shown as SEQ ID NO. 2.

2. The broad spectrum rabies virus-like particle antigen according to claim 1, wherein:

wherein the CVS strain rabies virus glycoprotein RVGP has 4 possible N-glycosylation sites Asn56, Asn266, Asn338 and Asn484, the first 3 is located in the ectodomain of the RVGP, and the 4 th is located in the cytoplasmic tail region of the RVGP;

the ectodomain of the CVS strain rabies virus glycoprotein RVGP contains B-cell epitopes, CLT-cell epitopes and Th-cell epitopes, and Asn56 and Asn266 are contained in the B-cell epitopes and Asn338 in the CTL-cell epitopes.

3. The method of designing the broad-spectrum rabies virus-like particle antigen as claimed in claim 1 or 2, comprising the steps of:

1) acquiring protein sequences of the CSV strains RVGP and RVMP by using an online website NCBI;

2) b cell epitopes in the RVGP antigen sequence are predicted by an online software ABCPredprediction server, so that the epitope regions are helped to be positioned and can be used for determining candidate vaccines;

3) potential CTL epitopes in RVGP were predicted by online software bioxgem. pacomplex and SYFPEITHI;

4) predicting potential Th epitopes in RVGP by on-line software RANKPEP;

5) predicting N-glycosylation sites in RVGP through an online software NetNGlyc 1.0 Server;

6) analyzing the change of epitope and tertiary structure of RVGP after glycosylation site mutation by bioinformatics software and PyMOL software in 2) -4);

7) obtaining 34 RVGP amino acid sequences separated from different regions in China from NCBI, comparing the obtained RVGP with the RVGP of a CSV strain by using DNAMAN software, determining the sequence consistency of the candidate RVGP and the RVGP of China origin, and further judging whether the candidate RVGP can be used as a candidate antigen of the ORV for Chinese animals.

4. A eukaryotic recombinant expression plasmid comprising the broad-spectrum rabies virus-like particle antigen of claim 1 or 2.

5. The method for constructing eukaryotic recombinant expression plasmid of claim 3, comprising the steps of:

1) respectively amplifying a promoter, SV40PA and fluorescent protein by taking eukaryotic plasmids as templates;

6. The method for constructing eukaryotic recombinant expression plasmid according to claim 5, wherein:

wherein, in the eukaryotic recombinant expression plasmid, the RVGP, the RVMP and the fluorescent protein all have independent promoters, ribosome binding sites and PloyA tails.

7. Eukaryotic expression system expressing the broad-spectrum rabies virus-like particle antigen according to claim 1 or 2, characterized in that said eukaryotic expression system is a HEK-293 cell or other mammalian cell capable of achieving post-translational modifications of mammalian carbohydrates.

8. A method for screening and identifying HEK-293 cells stably expressing the broad-spectrum rabies virus-like particle antigen of claim 1 or 2, comprising the steps of:

1) transfecting the eukaryotic plasmid containing RVGP, RVMP and EGFP according to claim 4 into HEK-293 cells and culturing the cells for selection of cell lines stably expressing RVLPs;

2) protein and mRNA levels of RVLPs were detected by WB and qPCR;

3) adopting 2mg/mL G418 to screen HEK-293 cell strains stably expressing broad-spectrum rabies virus-like particle antigens, replacing a fresh DMEM culture medium containing 2mg/mL G418 once every 2 days, culturing the positive cell strains, and after 14 days, enriching the positive monoclonal cell strains for 7 days to realize the amplification and passage of the positive monoclonal cell strains;

4) digesting the positive monoclonal cell strain, detecting the proportion and the average fluorescence intensity of EGFP positive cells by using a flow method, detecting the expression condition of RVLPs by using WB, selecting the cell strain with the EGFP positive cell proportion of more than 99 percent as a candidate stable cell strain, optimally selecting the cell strain with the highest average fluorescence intensity and RVLPs expression quantity as a stable cell strain, and naming the cell strain as HEK-293/RVLPs;

5) subjecting HEK-293/RVLPs to subculture, digesting the 10 th generation cells, inoculating a part of HEK-293/RVLPs into a confocal culture dish for culture, fixing the HEK-293/RVLPs with 4% paraformaldehyde at room temperature after 12h, staining nuclei for 10min with Hoechest 33342, washing with PBS, and observing the proportion of EGFP positive cells by using a laser confocal microscope; a part of cells are used for flow detection of EGFP positive cell proportion; inoculating a part of cells into a 24-well plate at different densities, and identifying the expression level of the target protein by WB; meanwhile, HEK-293/RVLPs cells are collected and used for cell ultrathin sections.

9. The method of claim 8, wherein:

in the step 3), when the positive monoclonal cell strain is cultured, replacing a fresh DMEM medium containing 2mg/mL G418 once every 2 days for 14 days;

when the positive monoclonal cell strain is enriched, cells are digested by pancreatin and diluted to 1/200 mu L, 200 mu L of the mixture is uniformly mixed and added into a 96-well plate, a fresh DMEM medium containing 1mg/mL is replaced once every 3 days, and the positive monoclonal cell strain is enriched for 7 days.